CN105663324A - Effective part group of yang-tonifying recuperating decoction and application thereof - Google Patents
Effective part group of yang-tonifying recuperating decoction and application thereof Download PDFInfo
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- CN105663324A CN105663324A CN201610042629.1A CN201610042629A CN105663324A CN 105663324 A CN105663324 A CN 105663324A CN 201610042629 A CN201610042629 A CN 201610042629A CN 105663324 A CN105663324 A CN 105663324A
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- ethanol
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- naringenin
- concentrated solution
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Abstract
The invention belongs to the technical field of medicine and relates to uses of various traditional Chinese medicine mixed compounds, in particular to application of an effective part group of yang-tonifying recuperating decoction in inducing mesenchymal stem cells to differentiate into nerve cells.The effective part group includes effective parts extracted from medicinal materials, including glucoside compounds, total alkaloids, Angelica sinensis polysaccharide, carthamin yellow and naringenin.Researches show that this effective part group can induce in vitro rat mesenchymal stem cells to differentiate into neuron-like cells and is applicable to drugs for treating nervous system diseases.
Description
Technical field
The invention belongs to pharmaceutical technology field, relate to the purposes of plurality of Chinese mixing cpd, be specifically related to a kind of Active Fraction of Buyang Huanwu Decoction group and the application in inducing bone mesenchymal mesenchymal stem cells into neurons breaks up thereof.
Background technology
Mesenchymal stem cells MSCs (bonemarrowmesenchymalstemcells, BMSCs) non-hematopoietic cell in myeloid tissue it is derived from, there is source easily obtain, do not limited by ethics, proliferation and differentiation ability widely, be easily isolated, cultivate, expand, purification, immunogenicity is weak, and immunologic tolerance is strong, outer-gene transfection efficiency is high, and can the advantage such as expression alien gene of stability and high efficiency. Therefore, MSCs becomes a kind of more satisfactory seed cell. Recent study report MSCs can be divided into neuron cell in vivo, in vitro, using basic fibrogenic cytokines (bFGF), 3-mercaptoethanol (BME), dimethyl sulfoxide (DMSO), butylated hydroxyanisole (BHA) or epithelical cell growth factor (EGF), retinoic acid (RA) etc. are derivant, it is possible to induce the BMSCs of rat and people for neuron cell. MSCs draws materials conveniently, easily obtains from autologous, immunological rejection will not occur after Hui Zhi; It is prone to separate in vitro, cultivate and amplification; The expression alien gene of in-vitro transfection rate high also energy stability and high efficiency, is therefore desirable seed cell. This provides new thinking for the treatment of nervous system injury and degenerative disease. Owing to MSCs is had very strong neurad cell induction effect by the synthetics such as DMSO, B-mercaptoethanol, but significantly toxicity, teratogenecity and carcinogenecity limit application. And Chinese medicine has based on the clinical experience of several thousand, its toxic and side effects is relatively small.
Chinese medicinal formulae BUYANG HUANWU TANG is used for treating Ischemic Stroke sequela clinically; modern pharmacology research finds that it has adjustment immunity, antiinflammatory regulating lipid metabolism, expansion of cerebral vascular; improving microcirculation, improve Hemorheology index, anticoagulation and inhibition thrombosis, free radical resisting, neuroprotective etc. act on. The survival rate of MSCs, rate of going back to the nest and differentiation rate can be improved with BUYANG HUANWU TANG intervention while transplanting MSCs in animal body. But Chinese medicinal formulae complicated component, the feature of its play a role and have " many because of, multiple-effect, Mutiple Targets ".
Summary of the invention
It is an object of the invention to provide a kind of Active Fraction of Buyang Huanwu Decoction group and the application in inducing bone mesenchymal mesenchymal stem cells into neurons breaks up thereof, this Active Fraction of Buyang Huanwu Decoction group can be applicable in treatment nervous system disease agent. This effective part group can promote that mesenchymal stem cells differentiation becomes neuron cell with inducing bone mesenchymal differentiation of stem cells.
Since we describe Chinese medicinal formulae BUYANG HUANWU TANG clinically for treating Ischemic Stroke sequela in background technology; modern pharmacology research finds that it has adjustment immunity, antiinflammatory regulating lipid metabolism, expansion of cerebral vascular; improving microcirculation, improve Hemorheology index, anticoagulation and inhibition thrombosis, free radical resisting, neuroprotective etc. act on.Through inventor's years of researches, test further, find to utilize sequential method to find the LD50 of each composition in BUYANG HUANWU TANG in early-stage Study, orthogonal design method is used to record its best compatibility dosage, it is developed into Active Fraction of Buyang Huanwu Decoction group (activeprincipleregionofBuyangHuanwudecoctionapr-BYHWD) by BUYANG HUANWU TANG side extracts each effective part group, and further study show that apr-BYHWD can induced rat mesenchymal stem cells differentiation be neuron cell in vitro, and its inducing effect is better than the former side of BUYANG HUANWU TANG, then inventor speculates that this effective part group can be applicable in treatment nervous system disease agent.
A kind of Active Fraction of Buyang Huanwu Decoction group provided by the invention application in inducing bone mesenchymal mesenchymal stem cells into neurons breaks up, described Active Fraction of Buyang Huanwu Decoction group is: glycosides compound, total alkaloids, Radix Angelicae Sinensis polysaccharide, Carthamus yellow and naringenin; The preparation method step of Active Fraction of Buyang Huanwu Decoction group is as follows:
(1) the separation purification of glycosides compound and naringenin effective site: take the Radix Astragali, Radix Paeoniae Rubra and Pheretima, adds ethanol, reflux, extract, filters, obtain medicinal residues A, standby; Filtrate recycling ethanol, adjusts and is equivalent to the concentrated solution of 0.5-3g medical material to every 1ml, and concentrated solution is washed with water after crossing macroporous resin column absorption, carries out eluting with ethanol afterwards, collects ethanol elution, concentrate drying, obtain glycosides compound and naringenin effective site;
(2) separation and Extraction of Carthamus yellow effective site: take Semen Persicae Flos Carthami, filter after boiling, merging filtrate, being concentrated into every 1ml and be equivalent to the concentrated solution of 0.5-3g medical material, concentrated solution is washed with water after crossing macroporous resin column absorption, afterwards with ethanol elution, collect eluent, concentrate drying, obtain Carthamus yellow effective site;
(3) extraction of Radix Angelicae Sinensis polysaccharide and total alkaloids effective site: get it filled slag A, Radix Angelicae Sinensis and Rhizoma Chuanxiong, boiling is filtered, obtain filtrate, at being concentrated into 40-60 DEG C, relative density is the clear paste of 1.02-1.10, adding ethanol makes alcohol content reach 45-60%, stand after stirring, filter, obtain precipitate B, filtrate flings to ethanol simultaneously, at being concentrated into 40-60 DEG C, relative density is the clear paste of 1.02-1.10, adding ethanol makes alcohol content reach 80-85%, stirring, rear standing, collect precipitate, dry to obtain Radix Angelicae Sinensis polysaccharide effective site, divide and take alcohol deposit fluid, fling to ethanol, add precipitate B to dissolve, add water to adjust to every 1ml and be equivalent to the concentrated solution of 0.5-3g medical material, after concentrated solution crosses the absorption of D101 macroporous resin column, wash with water, afterwards with ethanol elution, collect eluent, concentrate drying, obtain the effective site of total alkaloids.
A kind of Active Fraction of Buyang Huanwu Decoction group application in inducing bone mesenchymal mesenchymal stem cells into neurons breaks up, described Active Fraction of Buyang Huanwu Decoction group is: glycosides compound, total alkaloids, Radix Angelicae Sinensis polysaccharide, Carthamus yellow and naringenin; The preparation method of described Active Fraction of Buyang Huanwu Decoction group is:
(1) the separation purification of glycosides compound and naringenin effective site: take the Radix Astragali, Radix Paeoniae Rubra and Pheretima, adds ethanol percolate extraction, collects percolate, and medicinal residues A is standby; Percolate reclaims ethanol, adjust to every 1ml and be equivalent to the concentrated solution of 0.5-20g medical material, concentrated solution is added on polyamide column top, successively with pure water, 20%, 40%, 60%, 80% and 95% ethanol elution, eluent 100ml is as a fraction, the dynamic change of high-efficient liquid phase technique monitoring eluting fraction composition, the similar fraction of composition merges as a component, collect the fraction of astragaloside, peoniflorin, naringenin effective site, concentrate drying, obtain glycosides compound and naringenin effective site;
(2) separation and Extraction of Carthamus yellow and naringenin effective site: take Semen Persicae Flos Carthami, filter after boiling, merging filtrate, it is concentrated into every 1ml and is equivalent to the concentrated solution of 0.5-3g medical material, take concentrated solution and be added on polyamide column top, successively with pure water, 20%, 40%, 60%, 80% and 95% ethanol elution, eluent 100ml is as a fraction, the dynamic change of high-efficient liquid phase technique monitoring eluting fraction composition, collect the fraction of Carthamus yellow, naringenin, concentrate drying, obtains Carthamus yellow and naringenin effective site;
(3) extraction of Radix Angelicae Sinensis polysaccharide and total alkaloids effective site: get it filled slag A, Radix Angelicae Sinensis and Rhizoma Chuanxiong, filter after boiling, merging filtrate, at being concentrated into 40-60 DEG C, relative density is the clear paste of 1.02-1.10, adding ethanol makes alcohol content reach 45-60%, stirring is filtered after standing, obtain precipitate B, filtrate flings to ethanol simultaneously, at being concentrated into 40-60 DEG C, relative density is the clear paste of 1.02-1.10, adds ethanol and makes alcohol content reach 80-85%, and stirring stands, collect precipitate, dry to obtain Radix Angelicae Sinensis polysaccharide effective site; Separately take above-mentioned alcohol deposit fluid, fling to ethanol, add precipitate B to dissolve, add water to adjust to every 1ml and be equivalent to the concentrated solution of 0.5-3g medical material, take contracting liquid and be added on polyamide column top, successively with pure water, 20%, 40%, 60%, 80% and 95% ethanol elution, eluent 100ml is as a fraction, the dynamic change of high-efficient liquid phase technique monitoring eluting fraction composition, the similar fraction of composition merges as a component, collect the fraction of ligustrazine, ferulic acid effective site, concentrate drying, obtain total alkaloids effective site.
Above-mentioned Active Fraction of Buyang Huanwu Decoction group application in inducing bone mesenchymal mesenchymal stem cells into neurons breaks up, kind and the content of the medical material that the preparation of described Active Fraction of Buyang Huanwu Decoction group is used is: the Radix Astragali 23 66g, Radix Paeoniae Rubra 6 17g, Rhizoma Chuanxiong 6-10g, Radix Angelicae Sinensis 9 19g, Pheretima 9-17 gram, Flos Carthami 6 16g and Semen Persicae 9 20g.
The Active Fraction of Buyang Huanwu Decoction group of the present invention application in inducing bone mesenchymal mesenchymal stem cells into neurons breaks up, it is characterized in that, described Active Fraction of Buyang Huanwu Decoction group application in treatment nervous system disease agent, promote the medicinal usage of mesenchymal stem cells MSCs external stem cells into neuron-like cells direction conversion in preparation including this Active Fraction of Buyang Huanwu Decoction group, described mesenchymal stem cells MSCs directed differentiation is neuron cell.
Active Fraction of Buyang Huanwu Decoction group treats the application in nervous system disease agent, in the described process that mesenchymal stem cells MSCs directed differentiation is neuron cell, MEK-ERK and p38MAPK signal path participates in the process of this Active Fraction of Buyang Huanwu Decoction induction BMSCs neurad cell directional differentiation, and the blocking-up of MEK-ERK and p38MAPK signal path can suppress the Neural Differentiation of this effective ingredient in Chinese side combination induction BMSCs.
Another technical problem to be solved of the present invention is to provide and a kind of can promote the in-vitro directed effective ingredient in Chinese group being converted into neuron cell of mesenchymal stem cells MSCs. For this, the technical solution used in the present invention is: it adopts Active Fraction of Buyang Huanwu Decoction group as short cell differential agent, and inducing bone mesenchymal stem cell directional is divided into neuron cell.
Active Fraction of Buyang Huanwu Decoction group provided by the invention can promote that mesenchymal stem cells differentiation and inducing bone mesenchymal differentiation of stem cells become neuron cell, prepare into the medicine of nervous system disease, open up a new cell replacement therapy for neural multiple refractory disease.
Accompanying drawing explanation
Fig. 1 is the primary cell in the primary of BMSCs and Secondary Culture (× 100);
Fig. 2 is the 2nd generation cell in the primary of BMSCs and Secondary Culture (× 100);
Fig. 3 is the 3rd generation cell in the primary of BMSCs and Secondary Culture (× 100);
Fig. 4 is Flow cytometry rat BMSCs cell blank compared with control cells figure;
Fig. 5 is the BMSCs figure of Flow cytometry rat BMSCs cell FITC labelling CD90 positive expression;
Fig. 6 is the BMSCs figure of Flow cytometry rat BMSCs cell FITC labelling CD11 positive expression;
Fig. 7 is the BMSCs figure of Flow cytometry rat BMSCs cell FITC labelling CD45 positive expression;
Fig. 8 is the BMSCs of Western-Blots detection p-ERK protein expression electrophoresis pattern (1 blank cell, 2 negative control group, 3apr-BYHWD group) after BUYANG HUANWU TANG is induced;
Fig. 9 is the BMSCs of Western-Blots detection p-p38 protein expression electrophoresis pattern (1 blank cell, 2 negative control group, 3apr-BYHWD group) after BUYANG HUANWU TANG is induced;
Figure 10 is RT-PCR BMSCsNSEmRNA expression gel electrophoresis spectrum (the 1:apr-BYHWD matched group detecting induction after MAPK path blocks; 2:apr-BYHWD+SB203580 group; 3:apr-BYHWD+PD98059 group; M:Marker);
Figure 11 is BMSCsNSE protein expression gel electrophoresis spectrum (the 1:apr-BYHWD matched group induced after the MAPK path blocking-up of Western-Blots detection; 2:apr-BYHWD+SB203580 group; 3:apr-BYHWD+PD98059 group);
Figure 12 is RT-PCR BMSCsNestinmRNA expression gel electrophoresis spectrum (the 1:apr-BYHWD matched group detecting induction after MAPK path blocks; 2:apr-BYHWD+SB203580 group; 3:apr-BYHWD+PD98059 group; M:Marker);
Figure 13 is BMSCsNestin protein expression gel electrophoresis spectrum (the 1:apr-BYHWD matched group induced after the MAPK path blocking-up of Western-Blots detection; 2:apr-BYHWD+SB203580 group; 3:apr-BYHWD+PD98059 group).
Detailed description of the invention
The present invention is shown by embodiment research, and the combination of this Active Fraction of Buyang Huanwu Decoction can promote that mesenchymal stem cells MSCs directed differentiation in vitro is neuron cell. The present invention is described further in conjunction with Figure of description and embodiment.
Embodiment 1
A kind of Active Fraction of Buyang Huanwu Decoction group application in inducing bone mesenchymal mesenchymal stem cells into neurons breaks up, described Active Fraction of Buyang Huanwu Decoction group is: glycosides compound, total alkaloids, Radix Angelicae Sinensis polysaccharide, Carthamus yellow and naringenin; The preparation method step of Active Fraction of Buyang Huanwu Decoction group is as follows:
(1) the separation purification of glycosides compound and naringenin effective site: take Radix Astragali 60g, Radix Paeoniae Rubra 6g, Pheretima 9g, adds 5-15 times of 40%-70% ethanol, reflux, extract, 1-3 time, each 1-2h, filters, obtain medicinal residues A, standby; Filtrate recycling ethanol, adjusts and is equivalent to the concentrated solution of 0.5-3g medical material to every 1ml, and concentrated solution is washed with water after crossing macroporous resin column absorption, carry out eluting with 40-50% ethanol afterwards, collect ethanol elution, fling to ethanol, concentration, dry, obtain glycosides compound and naringenin effective site;
(2) separation and Extraction of Carthamus yellow effective site: take Semen Persicae 9g, Flos Carthami 9g, add 5-20 times of water, decoct 1-3 time, each 1-2 hour, filter, merging filtrate, being concentrated into every 1ml and be equivalent to the concentrated solution of 0.5-3g medical material, concentrated solution is washed with water after crossing macroporous resin column absorption, use 20-30% ethanol elution afterwards, collect eluent, fling to ethanol, concentration, dry, obtain Carthamus yellow effective site;
(3) extraction of Radix Angelicae Sinensis polysaccharide and total alkaloids effective site: get it filled slag A and Radix Angelicae Sinensis 9g, Rhizoma Chuanxiong 6g, add 5-20 times of water, decoct 1-3 time, each 1-2 hour, filter, merging filtrate, being concentrated at 40-60 DEG C relative density is the clear paste of 1.02-1.10, adding ethanol makes alcohol content reach 45-60%, stirring, stand 12-24 hour, filter, obtain precipitate B, filtrate flings to ethanol simultaneously, being concentrated at 40-60 DEG C relative density is the clear paste of 1.02-1.10, adding ethanol makes alcohol content reach 80-85%, stirring, stand 12-24 hour, collect precipitate, vacuum drying obtains Radix Angelicae Sinensis polysaccharide effective site, divide and take alcohol deposit fluid, fling to ethanol, add precipitate B to dissolve, add water to adjust to every 1ml and be equivalent to the concentrated solution of 0.5-3g medical material, after concentrated solution crosses the absorption of D101 macroporous resin column, wash with water, afterwards by 60-70% ethanol elution macroporous resin column, collect eluent, fling to ethanol, concentrate drying, obtain total alkaloids effective site.
Embodiment 2
A kind of Active Fraction of Buyang Huanwu Decoction group application in inducing bone mesenchymal mesenchymal stem cells into neurons breaks up, described Active Fraction of Buyang Huanwu Decoction group is: glycosides compound, total alkaloids, Radix Angelicae Sinensis polysaccharide, Carthamus yellow and naringenin; The preparation method step of Active Fraction of Buyang Huanwu Decoction group is as follows:
(1) the separation purification of glycosides compound and naringenin effective site: take Radix Astragali 60g, Radix Paeoniae Rubra 6g, Pheretima 9g, adds 5-15 times of 40%-70% ethanol, through seepage pressure effects, collects percolate, and medicinal residues A is standby; Percolate reclaims ethanol, adjust to every 1ml and be equivalent to the concentrated solution of 0.5-20g medical material, concentrated solution is added on polyamide column top, successively with pure water, 20%, 40%, 60%, 80% and 95% ethanol elution, eluent 100ml is as a fraction, the dynamic change of high-efficient liquid phase technique monitoring eluting fraction composition, the similar fraction of composition merges as a component, collect the fraction of astragaloside, peoniflorin, naringenin effective site, concentrate drying, obtain glycosides compound and naringenin effective site;
(2) separation and Extraction of Carthamus yellow and naringenin effective site: take Semen Persicae 9g, Flos Carthami 9g, add water about 5-20 times, decoct 1-3 time, each 1-2 hour, filter, merging filtrate, it is concentrated into every 1ml and is equivalent to the concentrated solution of 0.5-3g medical material, take concentrated solution and be added on polyamide column top, successively with pure water, 20%, 40%, 60%, 80% and 95% ethanol elution, eluent 100ml is as a fraction, the dynamic change of high-efficient liquid phase technique monitoring eluting fraction composition, collect flavochrome, the fraction of naringenin, concentrate drying, obtain Carthamus yellow and naringenin effective site,
(3) extraction of Radix Angelicae Sinensis polysaccharide and total alkaloids effective site: get it filled slag A and Radix Angelicae Sinensis 9g, Rhizoma Chuanxiong 6g, add water about 5-20 times, decoct 1-3 time, each 1-2 hour, filter, merging filtrate, at being concentrated into 40-60 DEG C, relative density is the clear paste of 1.02-1.10, adding ethanol makes alcohol content reach 45-60%, stirring, stand 12-24 hour, filter, obtain precipitate B, filtrate flings to ethanol simultaneously, at being concentrated into 40-60 DEG C, relative density is the clear paste of 1.02-1.10, adding ethanol makes alcohol content reach 80-85%, stirring, stand 12-24 hour, collect precipitate, dry to obtain Radix Angelicae Sinensis polysaccharide effective site, separately take above-mentioned alcohol deposit fluid, fling to ethanol, add precipitate B to dissolve, add water to adjust to every 1ml and be equivalent to the concentrated solution of 0.5-3g medical material, take contracting liquid and be added on polyamide column top, successively with pure water, 20%, 40%, 60%, 80% and 95% ethanol elution, eluent 100ml is as a fraction, the dynamic change of high-efficient liquid phase technique monitoring eluting fraction composition, the similar fraction of composition merges as a component, collects the fraction of ligustrazine, ferulic acid effective site, flings to ethanol, concentrate drying, obtains total alkaloids effective site.
Embodiment 3
This Active Fraction of Buyang Huanwu Decoction group is at the induction Analytical Chemical Experiment research promoting mesenchymal stem cells MSCs directed differentiation to be in vitro neuron cell and MAPK function analysis wherein.
Material:
Animal: 6～8 week old cleaning grade male SD rats 10, weight (130 ± 20) g, purchased from Shanghai Slac Experimental Animal Co., Ltd., animal credit number: SCXK(Shanghai) 2009-0004. Animal House temperature 25 DEG C, humidity 60%. The strict middle pertinent regulations with reference to " about the kind treatment laboratory animal directiveness suggestion " of Ministry of Science & Technique of PRC's promulgation of the operation of animal are carried out by rat experiment.
Medicine: the Radix Astragali (Classification system: Leguminosae) 60g, Radix Paeoniae Rubra (Classification system: PaeonialactifloraPall.) 6g, Rhizoma Chuanxiong (Classification system: RhizomaChuanxiong) 6g, Radix Angelicae Sinensis (formal name used at school: Angelicasinensis) 9g, Pheretima [Classification system Pheretimaaspergillum (E.Perrier)) 9g, Semen Persicae [Classification system: Prunuspersica(L.) Batsch)] 9g, Flos Carthami (latin name: CarthamustinctoriusL.) 9g. By its proportion of composing, adopt the method in embodiment 1 or embodiment 2 to prepare effective part group and (prepare former side's extracting solution with decoction alcohol precipitation method, acid-base precipitation, resinbed analysis method is adopted to extract effective site former side's extracting solution again, each effective site separates completely, each other without mixing, purity is more than 70%, press the content of its Quality Control thing of following standard test with high pressure liquid chromatography and chemical analysis method, wherein in alkaloid, ligustrazine content is 9.76mg/g alkaloid) it is made into desired concn with serum-free L-DMEM culture medium.
Method:
(1) separation of BMSCs, cultivation and qualification: male SD rat cervical dislocation is put to death, soaks 10min in 75% ethanol. Quickly take tibia and femur under aseptic condition, excise two ends. Aspirate DMEM liquid with No. 8 syringe needles and repeatedly rinse medullary cavity, collect cell. Add containing 10% hyclone (Hyclone company, the U.S.) DMEM culture medium (GIBCO company, the U.S.), repeatedly blow and beat, with 1 × 10 after cell counting9/ mL is inoculated in floor space 50cm2In culture bottle, 37 DEG C, saturated humidity, be the CO of 0.05 containing volume fraction2Incubator (THERMOFORMA, the U.S.) is cultivated. Abandon suspension after 48h, change liquid first. Every 2d changes liquid 1 time later, and the hematopoietic cell of suspension growth is discarded with changing liquid, leaves the BMSCs of adherent growth. About treat that cell closes in bottle undermelting and basically reach about 80%, 0.25% trypsin SIGMA company, U.S.) collect cell after digestion. Repeat aforesaid operations, repeatedly reach P3Generation. Take the 3rd generation BMSCs, after 0.25% trypsinization, be configured to cell suspension, 1%BSA confining liquid 10min, PBS washs 3 times, add FITC fluorescently-labeled anti-CD11b, CD45, CD90 antibody (1:1000) 0.5mL(SERATEC company, Germany), matched group adds PBS, hatch 30min for 4 DEG C, 1500r/min is centrifuged 5min, PBS and rinses 3 times, fixes with 1% paraformaldehyde, FACScan flow cytometer (BD company, the U.S.) is adopted to check cell surface antigen. With CD45, CD11b positive rate lower than 5%, higher than 95%, CD90 positive rate identifies that gained cell is highly purified BMSCs.
(2) expression of phosphorylated protein ERK, p-38 in BMSCs after apr-BYHWD induction:
The BMSCs reaching for the 4th generation is tested.Experiment point 3 groups.
1. laboratory observation group: add 0.039g/Lapr-BYHWD in rat BMSCs culture fluid.
2. negative control group: add in rat BMSCs culture fluid and 1. group equivalent DMEM.
3. Normal group: add in rat BMSCs culture fluid with 1. group equivalent containing 10% hyclone DMEM.
3 groups all put incubator hatch 24h after, remove the culture fluid of each group, wash 2 times with DMEM culture medium, then continue to cultivate by above-mentioned condition of culture, every 3d changes liquid 1 time. After induction differentiation 7 days, 14 days, 28 days, take out each group of BMSCs respectively, make phosphorylated protein ERK, p-38 and identify.
(3) apr-BYHWD is induced the impact of BMSCs Neural Differentiation by MAPK signal path:
The BMSCs reaching for the 4th generation is randomly divided into apr-BYHWD matched group, apr-BYHWD+PD98059 group, apr-BYHWD+SB203580 group.
1. apr-BYHWD matched group: add containing apr-BYHWD(0.39%) induction broth 3ml cultivates after BMSCs hatches 24h, removes culture fluid, washs 2 times with DMEM culture medium, then continues to cultivate by above-mentioned condition of culture, and every 3d changes liquid 1 time. After induction differentiation 7 days, 14 days, 28 days, carry out neural-like cells qualification.
2. apr-BYHWD+PD98059 group: add containing apr-BYHWD(0.39%) induction broth 3ml cultivates BMSCs, add apr-BYHWD induce liquid be simultaneously introduced 30 μ l1000 μm of ol/LPD98059 (MEK-ERK signal pathway inhibitor, SIGMA company, the U.S.), PD98059 is made to be diluted to 10 μm of ol/L, after hatching 24h, remove culture fluid, wash 2 times with DMEM culture medium, continue to cultivate by above-mentioned condition of culture again, every 3d changes liquid 1 time, and induction carries out neural-like cells qualification after breaking up 7 days, 14 days, 28 days.
3. apr-BYHWD+SB203580 group: add containing apr-BYHWD(0.39%) induction broth 3ml cultivates BMSCs, add apr-BYHWD induce liquid be simultaneously introduced l1500 μm of ol/LSB203580 of 30 μ (p38MAPK signal transduction pathway inhibitor, SIGMA company, the U.S.), SB203580 is made to be diluted to 15 μm of ol/L, after hatching 24h, remove culture fluid, wash 2 times with DMEM culture medium, continue to cultivate by above-mentioned condition of culture again, every 3d changes liquid 1 time, and induction carries out neural-like cells qualification after breaking up 7 days, 14 days, 28 days.
(4) RT-PCR detects the expression of cell Nestin and NSEmRNA:
By the BMSCs of different time sections after induction with being configured to cell suspension about 1 × 10 after 0.25% trypsinization7/ mL, Trizol(Invitrogen company, Canada) cell lysis, extracts cell total rna. After its integrity of electroresis appraisal, DU640 type nucleic acid-protein analyser (Backman company, the U.S.) is utilized to measure A260With A280Ratio, and calculate its concentration, if β-actinmRNA is internal reference. With the NCBIGenbank primer sequence announced, use PrimerPremier5.0(PremierBiosoft company, the U.S.) it is designed and carries out Blast checking, and synthesized by Invitrogen company.
PCR primer sequence and amplification purpose fragment:
RT-PCR adopts two step method to carry out, and grads PCR instrument (Bio-rad, the U.S.) reverse transcription is 65 DEG C, 5min first, 42 DEG C, 60min synthesizes cDNA then 70 DEG C of degeneration 5min, then PCR carries out 35 cyclic amplifications (Nestin:94 DEG C, 30s, 54 DEG C, 1min, 72 DEG C, 1min; NSE:94 DEG C, 30s, 56 DEG C, 1min, 72 DEG C, 1min), last 72 DEG C extend 10min.After PCR terminates, with 2% agarose gel electrophoresis detection amplification. Utilize the absorbance ratio of gel imaging system (UVItec company, Britain) scanning analysis Nestin, NSEmRNA and internal reference β-actinmRNA. Experiment repeats 3 times.
(5) Western-blots detects phosphorylated CREB, phosphorylation p-38, Nestin and NSE protein content:
The BMSCs of induction different time sections is extracted total protein, and BCA method measures protein concentration; SDS-PAGE electrophoresis and transferring film; Put the PVDF after electrotransfer by anti-for rabbit rat p-ERK, p-p-38, Nestin and NSE monoclonal antibody (SantaCruzBiotechnology, SantaCruz, CA, USA) it is diluted to debita spissitudo with TBST by 1:300, hybridization bag is used after carrying out overnight incubation, to prepare the goat anti-rabbit igg (SantaCruzBiotechnology of HRP labelling, SantaCruz, CA, USA) after diluent (1:3000) incubated at room temperature 2h, carry out ECL chemiluminescence reaction, in darkroom, X-ray film is exposed 1～10min, developed fixing after, it determines result. On gel imaging system, Image-ProPlus6.0(MediaCybernetics company is used in scanning, USA) software carries out cumulative absorbance value analysis, with destination protein absorbance and β-actin (SantaCruzBiotechnology, SantaCruz, CA, USA) ratio of absorbance represents the expression of destination protein.
(6) statistical analysis: continuous variable represents with mean ± SD, multi-group data adopts one factor analysis of variance, homogeneity test of variance and normal distribution to carry out variance analysis after assessing, and compares with the inspection of LSD-T method between group between two, as P < 0.05, difference significance. All data use SPSS17.0(SPSS company, Chicago,U.S) carry out statistical analysis.
Result
(1) the BMSCs form of original cuiture
The BMSCs cell of original cuiture, rounded under mirror, not of uniform size, suspension cell is intensive, starts adherent after 4～6h, and after 24h, major part BMSCs is all adherent, changes liquid first and discard non-attached cell after 48h. After 3～4d, cell is colony growth, converges in flakes gradually, and its form is changed to short fusiformis or tip-like more, it is seen that kernel, at the bottom of 7～10d is paved with bottle substantially.
After going down to posterity, cell is completely adherent in 24h, and the 3rd generation cell volume relatively primary cell increases, major part fusiformis or fibrous arrangement, and endochylema is transparent, and karyon is substantially (as shown in Figure 1, Figure 2, Figure 3 shows).
(2) qualification of BMSCs surface antigen
Application flow cytometer checks that BMSCs surface antigen CD90 positive cell rate all reaches more than 98%, and CD11b and CD45 positive rate is less than 2%(such as shown in Fig. 4, Fig. 5, Fig. 6, Fig. 7), showed cell homogeneity is better, and purity is higher.
(3) expression of phosphorylated protein ERK, p-38 in BMSCs after Active Fraction of Buyang Huanwu Decoction induction
In apr-BYHWD group, the 28th day ERK protein phosphorylation level is higher than blank group and negative control group, and the 28th day level higher than 7 days and 14 days, its difference has statistical significance (P < 0.05, Fig. 8 table 1); Other groups have no significant difference. In apr-BYHWD group, 7 days, 14 days, 28 days horizontal p-38 phosphorylated protein levels are higher than blank group and negative control group (P < 0.05, Fig. 9 table 1), wherein have no significant difference at 7 days, 14 days, 28 days after its induction.
(4) apr-BYHWD is induced the impact that after BMSCs, NSE expresses by MAPK path blocker
In apr-BYHWD matched group, 7 days, 14 days, 28 days all visible NSEmRNA express, in apr-BYHWD+SB203580 group, express weak compared with apr-BYHWD matched group when 14 days, 28 days, its difference has statistical significance (P < 0.05, Figure 10, table 2), within 7 days, 14 days, 28 days in apr-BYHWD+PD98059 group, express and be below apr-BYHWD comparison (P < 0.05, Figure 10, table 2).The BMSCsNSE protein expression that the MAPK path of Western-Blots detection is induced after blocking, within 7 days, 14 days, 28 days in apr-BYHWD+SB203580 group and apr-BYHWD+PD98059 group, express and be below apr-BYHWD comparison (P < 0.05, Figure 11, table 2) express basically identical with NSEmRNA.
(5) apr-BYHWD is induced the impact that after BMSCs, Nestin expresses by MAPK path blocker
In apr-BYHWD matched group, 7 days, 14 days, 28 days all visible NestinmRNA express, in apr-BYHWD+SB203580 group, 28 days time have a small amount of expression, its expression is weak compared with apr-BYHWD matched group, its difference has statistical significance (P < 0.05, Figure 12 table 3), within 7 days, 14 days, 28 days in apr-BYHWD+PD98059 group, it is showed no expression. The BMSCsNestin protein expression that the MAPK path of Western-Blots detection is induced after blocking, apr-BYHWD+SB203580 group and apr-BYHWD+PD98059 group are showed no expression when 7 days, 14 days, when 28 days, expression is below apr-BYHWD comparison (P < 0.05, Figure 13 table 3) and NestinmRNA and expresses basically identical on a small quantity.
BMSCs differentiating into nerve cells under specific environment (inductive condition) is confirmed, one of research focus being currently biological study of its directed differentiation mechanism. In above-mentioned experiment, we have looked first at after apr-BYHWD induces MAPK phosphorylated protein expression in BMSCs, found that increasing, peak occurred when the 14th day, 28 days occur in p-p38 phosphorylation level during after induction 7 days. Then there is increasing of relatively matched group when 28 days in p-ERK. Prompting apr-BYHWD has activated MAPK path, thus performing itself and cell proliferation and differentiation correlation function. The activation of p38MAPK approach is earlier in MEK-ERK approach.
Next we select cell-signaling pathways blocker PD98059 and SB203580 2 kinds different to block MEK-ERK and p38MAPK signal pathway respectively. Result is pointed out, NSE after blocking-up MEK-ERK path, within 7 days, 14 days, 28 days, all occurs the decline expressed at gene level and protein level, and Nestin had no the expression of mRNA, the expression of a small amount of albumen detected when 28 days. After blocking p38MAPK approach, NSEmRNA expressed minimizing when 14 days, 28 days, and its albumen all expressed minimizing when 7,14,18 days. NestinmRNA and albumen were all not detected by 7 days, 14 days expressing, and expressed on a small quantity when 28 days. Illustrate that MAPK path induces BMSCs be activated by and affect the expression of NSE, Nestin at apr-BYHWD. Prompting MEK-ERK and p38MAPK signal path participates in apr-BYHWD and leads BMSCs nerve orientation atomization.
Conclusion: Active Fraction of Buyang Huanwu Decoction side provided by the invention can induce BMSCs Differentiation into Neuron-like Cells in vitro, MEK-ERK and p38MAPK signal path participates in apr-BYHWD and induces BMSCs neurad cell directional atomization, and blocking of MEK-ERK and p38MAPK signal path suppresses the apr-BYHWD Neural Differentiation inducing BMSCs.
Claims (5)
1. the Active Fraction of Buyang Huanwu Decoction group application in inducing bone mesenchymal mesenchymal stem cells into neurons breaks up, it is characterized in that, described Active Fraction of Buyang Huanwu Decoction group is: glycosides compound, total alkaloids, Radix Angelicae Sinensis polysaccharide, Carthamus yellow and naringenin; The preparation method step of Active Fraction of Buyang Huanwu Decoction group is as follows:
(1) the separation purification of glycosides compound and naringenin effective site: take the Radix Astragali, Radix Paeoniae Rubra and Pheretima, adds ethanol, reflux, extract, filters, obtain medicinal residues A, standby;Filtrate recycling ethanol, adjusts and is equivalent to the concentrated solution of 0.5-3g medical material to every 1ml, and concentrated solution is washed with water after crossing macroporous resin column absorption, carries out eluting with ethanol afterwards, collects ethanol elution, concentrate drying, obtain glycosides compound and naringenin effective site;
(2) separation and Extraction of Carthamus yellow effective site: take Semen Persicae Flos Carthami, filter after boiling, merging filtrate, being concentrated into every 1ml and be equivalent to the concentrated solution of 0.5-3g medical material, concentrated solution is washed with water after crossing macroporous resin column absorption, afterwards with ethanol elution, collect eluent, concentrate drying, obtain Carthamus yellow effective site;
(3) extraction of Radix Angelicae Sinensis polysaccharide and total alkaloids effective site: get it filled slag A, Radix Angelicae Sinensis and Rhizoma Chuanxiong, boiling is filtered, obtain filtrate, at being concentrated into 40-60 DEG C, relative density is the clear paste of 1.02-1.10, adding ethanol makes alcohol content reach 45-60%, stand after stirring, filter, obtain precipitate B, filtrate flings to ethanol simultaneously, at being concentrated into 40-60 DEG C, relative density is the clear paste of 1.02-1.10, adding ethanol makes alcohol content reach 80-85%, stirring, rear standing, collect precipitate, dry to obtain Radix Angelicae Sinensis polysaccharide effective site, divide and take alcohol deposit fluid, fling to ethanol, add precipitate B to dissolve, add water to adjust to every 1ml and be equivalent to the concentrated solution of 0.5-3g medical material, after concentrated solution crosses the absorption of D101 macroporous resin column, wash with water, afterwards with ethanol elution, collect eluent, concentrate drying, obtain the effective site of total alkaloids.
2. the Active Fraction of Buyang Huanwu Decoction group application in inducing bone mesenchymal mesenchymal stem cells into neurons breaks up, it is characterised in that the preparation method of described Active Fraction of Buyang Huanwu Decoction group is:
(1) the separation purification of glycosides compound and naringenin effective site: take the Radix Astragali, Radix Paeoniae Rubra and Pheretima, adds ethanol percolate extraction, collects percolate, and medicinal residues A is standby; Percolate reclaims ethanol, adjust to every 1ml and be equivalent to the concentrated solution of 0.5-20g medical material, concentrated solution is added on polyamide column top, successively with pure water, 20%, 40%, 60%, 80% and 95% ethanol elution, eluent 100ml is as a fraction, the dynamic change of high-efficient liquid phase technique monitoring eluting fraction composition, the similar fraction of composition merges as a component, collect the fraction of astragaloside, peoniflorin, naringenin effective site, concentrate drying, obtain glycosides compound and naringenin effective site;
(2) separation and Extraction of Carthamus yellow and naringenin effective site: take Semen Persicae Flos Carthami, filter after boiling, merging filtrate, it is concentrated into every 1ml and is equivalent to the concentrated solution of 0.5-3g medical material, take concentrated solution and be added on polyamide column top, successively with pure water, 20%, 40%, 60%, 80% and 95% ethanol elution, eluent 100ml is as a fraction, the dynamic change of high-efficient liquid phase technique monitoring eluting fraction composition, collect the fraction of Carthamus yellow, naringenin, concentrate drying, obtains Carthamus yellow and naringenin effective site;
(3) extraction of Radix Angelicae Sinensis polysaccharide and total alkaloids effective site: get it filled slag A, Radix Angelicae Sinensis and Rhizoma Chuanxiong, filter after boiling, merging filtrate, at being concentrated into 40-60 DEG C, relative density is the clear paste of 1.02-1.10, adding ethanol makes alcohol content reach 45-60%, stirring is filtered after standing, obtain precipitate B, filtrate flings to ethanol simultaneously, at being concentrated into 40-60 DEG C, relative density is the clear paste of 1.02-1.10, adds ethanol and makes alcohol content reach 80-85%, and stirring stands, collect precipitate, dry to obtain Radix Angelicae Sinensis polysaccharide effective site; Separately take above-mentioned alcohol deposit fluid, fling to ethanol, add precipitate B to dissolve, add water to adjust to every 1ml and be equivalent to the concentrated solution of 0.5-3g medical material, take contracting liquid and be added on polyamide column top, successively with pure water, 20%, 40%, 60%, 80% and 95% ethanol elution, eluent 100ml is as a fraction, the dynamic change of high-efficient liquid phase technique monitoring eluting fraction composition, the similar fraction of composition merges as a component, collect the fraction of ligustrazine, ferulic acid effective site, concentrate drying, obtain total alkaloids effective site.
3. the Active Fraction of Buyang Huanwu Decoction group according to claim 1 and 2 application in inducing bone mesenchymal mesenchymal stem cells into neurons breaks up, it is characterized in that, kind and the content of the medical material that the preparation of described Active Fraction of Buyang Huanwu Decoction group is used is: the Radix Astragali 23 66g, Radix Paeoniae Rubra 6 17g, Rhizoma Chuanxiong 6-10g, Radix Angelicae Sinensis 9 19g, Pheretima 9-17 gram, Flos Carthami 6 16g and Semen Persicae 9 20g.
4. the Active Fraction of Buyang Huanwu Decoction group according to claim 1 and 2 application in inducing bone mesenchymal mesenchymal stem cells into neurons breaks up, it is characterized in that, described Active Fraction of Buyang Huanwu Decoction group application in treatment nervous system disease agent, promote the medicinal usage of mesenchymal stem cells MSCs external stem cells into neuron-like cells direction conversion in preparation including this Active Fraction of Buyang Huanwu Decoction group, described mesenchymal stem cells MSCs directed differentiation is neuron cell.
5. the Active Fraction of Buyang Huanwu Decoction group according to claim 4 application in treatment nervous system disease agent, it is characterized in that, described mesenchymal stem cells differentiation is in the process of neuron cell, MEK-ERK and p38MAPK signal path participates in the process of this Active Fraction of Buyang Huanwu Decoction induction BMSCs neurad cell directional differentiation, and the blocking-up of MEK-ERK and p38MAPK signal path can suppress the Neural Differentiation of this effective ingredient in Chinese side combination induction BMSCs.
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Non-Patent Citations (4)
| Title |
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| JINGHUI ZHENG, JIAN LIANG, XIN DENG: "Mitogen activated protein kinase signaling pathways participate in the active principle region of Buyang Huanwu decoction-induced differentiation of bone marrow mesenchymal stem cells", 《NEURAL REGENERATION RESEARCH》 * |
| JINGHUI ZHENG，YI WAN, JIANHUAI CHIT: "The active principle region of Buyang Huanwu decoction induced differentiation of bone marrow-derived mesenchymal stem cells into neural-like cells: Superior effects over original formula of Buyang Huanwu decoction", 《NEURAL REGENERATION RESEARCH》 * |
| 张运克，张振强，高峰等: "补阳还五汤诱导大鼠骨髓间充质干细胞向", 《2013年度中华中医药学会科学技术获奖奖项项目简介》 * |
| 邓展生，张璇，邹冬青等: "骨碎补有效成分柚皮疳对人骨髓间充质", 《湖南学院学报》 * |
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